A synapse is comprised of two compartments: presynaptic (the axon terminal) and postsynaptic (the target membrane). During the formation of a synapse, the pre- and postsynaptic compartments both differentiate. Much has been learned about postsynaptic differentiation, but the molecular machinery of presynaptic differentiation remains largely unknown. Identification of the genes that are important for presynaptic development will help fill this gap and facilitate the understanding of brain functions. In C. elegans, presynaptic differentiation requires the serine/threonine kinase Sad-1: synaptic vesicle clustering is decreased in the Sad-1 mutant and overexpression of Sad-1 causes vesicles to mislocalize. This proposed study is aimed at elucidating the functions of the murine orthologues of Sad-1 in presynaptic differentiation. There are two orthologues of Sad-1 in mice, Sad-1A and B. Double knockout mice, recently generated in our lab, are paralyzed and die at birth. The first aim is to look at Sad-1 tissue and cellular distribution by antibody staining and in situ hybridization. This will provide information about where Sad-1 is functioning. The second aim is to characterize the phenotype of the Sad-1 knockout mice. The formation of synapses as well as other aspects of neurodevelopment will be examined in tissues or isolated neurons from knockout mice. The third aim is to generate CRE mediated conditional Sad-1 knockout. This will help the animals live past birth and allow observation of postnatal synaptogenesis [unreadable] [unreadable]